The DNA topoisomerases are a family of enzymes that play multiple roles in the maintenance and propagation of the genomes of both prokaryotes and eukaryotes. Compounds that act as effective cellular inhibitors or “poisons” of topoisomerases act as cytotoxic agents through induction of DNA damage.
Topoisomerases have been reviewed in, for example, Wigley, D. B. (1995) Ann. Rev. Biophys. Biomolec. Struct. 24: 185–208. Type I DNA topoisomerases (EC 5.99.1.2; also known as relaxing enzyme, untwisting enzyme, swivelase, nicking-closing enzyme, and omega-protein) can convert one topological isomer of DNA into another. In particular, these topoisomerases can relax superhelical turns in DNA, interconvert simple and knotted rings of single-stranded DNA, and intertwist single-stranded rings of complementary sequences. Additionally, the type I topoisomerases act by catalyzing the transient breakage of DNA, one strand at a time, and the subsequent rejoining of the strands. In the process of breaking the strand, a type I topoisomerase (or topoisomerase I or topo I) simultaneously forms a topoisomerase-DNA link in which the hydroxyl group of a tyrosine residue is joined to a 5′-phosphate on DNA, at one end of the enzyme-severed DNA strand.
Similarly, type II DNA topoisomerases (EC 5.99.1.3; also known as DNA topoisomerase II and DNA gyrase) can change the topology of double-stranded DNA molecules, causing, for example, the relaxation of supercoiled DNA molecules, catenation, decatenation, knotting and unknotting of circular DNA (for a review, see Watt and Hickson (1994) Biochem. J. 303: 681–695). Type II topoisomerases act by a concerted breakage and reunion activity involving both strands of the DNA duplex. This activity is absolutely required for DNA replication and transcription.
Still other types of topoisomerases have been identified, including a cDNA encoding human DNA topoisomerase III (Hanai, et al. Proc. Nat. Acad. Sci. 93: 3653–3657 (1996)) which is commonly deleted in patients with the Smith-Magenis syndrome (Elsea, et al., Am. J. Med. Genet. 75: 104–108 (1998)). DNA topoisomerase III protein is homologous to the E. coli DNA topoisomerase I subfamily of enzymes, but shares no significant sequence homology with eukaryotic DNA topoisomerase I. Topoisomerase III catalyzes the reduction of supercoils in highly negatively supercoiled DNA.
Currently, topoisomerase inhibitors are classified into two general types. The class designated as “poisons” have in common the property of causing “trapping” of the target topoisomerase in the form of a covalent complex with the nucleic acid substrate. The “non-poison” class inhibits the enzymatic activity of the topoisomerase without specific effects on steps of the catalytic cycle that involve formation or resolution of the enzyme-DNA covalent intermediate. Of the DNA topoisomerase inhibitors currently used as clinical antibiotic or antineoplastic agents, the “poisons” seem to be most effective, probably because such compounds result in the accumulation of irreversible genotoxic damage in target cells.
Additionally, there are currently two type I topoisomerase (“Topo I”) poisons approved for the treatment of cancer in the United States—topotecan and irinotecan (Abang, et al., Seminars in Hematology 35: 13–21 (1998), Arbuck, et al., Seminars in Hematology 35: 3–12 (1998), Thompson, et al., Biochimica et Biophysica Acta. 1400: 301–19 (1998)), both of which are derivatives of camptothecin. These two agents have demonstrated clinical activity, but in rather distinct tissue settings (Abang, et al., Seminars in Hematology 35: 13–21 (1998), Arbuck, et al., Seminars in Hematology 35: 3–12 (1998), Thompson, et al., Biochimica et Biophysica Acta. 1400: 301–19 (1998)). The use of these FDA-approved Topo I poisons is further restricted by tumor resistance and dose limiting toxicities (Abang, et al., Seminars in Hematology 35: 13–21 (1998) Arbuck, et al., Seminars in Hematology 35: 3–12 (1998), Cersosimo, et al., The Annals of Pharmacotherapy 32: 1334–43 (1998), Dingemans, et al., Biochimica et Biophysica Acta. 1400: 275–88 (1998), Kollmannsberger, et al., Oncology, 56: 1–12 (1999), Larsen, et al., Biochimica et Biophysica Acta. 1400: 257–74 (1998), Slichenmyer, et al., Chemotherapy & Pharmacology 34 Suppl: S53–7 (1994)). Significant interest has now developed in identifying additional compounds that target Topo I and that may overcome some of these limitations. This has resulted in the description of a number of Topo I-targeted agents including camptothecin derivatives (Pommier, et al., Biochimie 80: 255–270 (1998)), molecules that bind DNA by intercalation (Fukasawa, et al., International Journal of Cancer 75: 145–50 (1998), Gatto, et al., Cancer Research 56: 2795–800 (1996), Guano, et al., Molecular Pharmacology 56: 77–84 (1999), Pilch, et al., Biochemistry 36: 12542–53 (1997). Yoshinari, et al., Cancer Research 55: 1310–5 (1995)) or that bind to the minor groove in DNA (Pilch, et al., Biochemistry 36: 12542–53 (1997), Bridewell, et al., Oncology Research 9: 535–42 (1997), Chen, et al., Academy of Sciences of the United States of America 90: 8131–5 (1993), Chen, et al., Cancer Research 53: 1332–7 (1993), Martinez, et al., National Academy of Sciences of the United States of America 96: 3496–501 (1999), Nitiss, et al., Cancer Research 57: 4564–9 (1997), Sim, et al., Biochemistry 36: 13285–91 (1997), Takebayashi, et al., Proceedings of the National Academy of Sciences of the United States of America 96: 7196–201 (1999), Xu, et al., Biochemistry 37: 3558–66 (1998)), and others (Kohlhagen, et al, Molecular Pharmacology 54: 50–8 (1998)). Additionally, compounds that are less selective and act as both Topo I and Topo II poisons (Leteurtre, et al., Journal of Biological Chemistry 269: 28702–7 (1994), Poddevin, et al., Molecular Pharmacology 44: 767–74 (1993), Riou, et al., Cancer Research 53: 5987–93 (1993), Yamashita, et al., Biochemistry 30: 5838–45 (1991)) have been described.
Camptothecin has been shown to act by stabilizing an otherwise transient reaction intermediate between Topo I and DNA (Abang, et al., Seminars in Hematology 35: 13–21 (1998), Arbuck, et al., Seminars in Hematology 35: 3–12 (1998). Pommier, et al., Biochimie 80: 255–270 (1998)). In these trapped complexes Topo I is covalently attached to the 3′ end of a single strand break. Removal of camptothecin results in rapid religation of the break by Topo I and averts toxicity. Toxicity occurs during S-phase when a replication fork encounters a trapped complex and converts it into a double strand break (DSB, D'Arpa, et al., Cancer Research 50: 6919–24 (1990), Hsiang, et al., Cancer Research 49: 5077–82 (1989), Holm, et al., Cancer Research 49: 6365–8 (1989)). Consequently, the toxicity of camptothecin and its derivatives show a strong dependency on the time of exposure in vitro (Cheng, et al., Oncology Research 6: 269–79 (1994)) and in vivo (Haas, et al., Cancer Research 54: 1220–6 (1994), Houghton, et al., Cancer Chemotherapy & Pharmacology 36: 393–403 (1995), Rodman, et al., Journal of Clinical Oncology 5: 1007–14 (1987)). Furthermore, in tissue culture cell lines camptothecin toxicity can be inhibited by co-treatment with DNA synthesis inhibitors (D'Arpa, et al., Cancer Research 50: 6919–24 (1990), Hsiang, et al., Cancer Research 49: 5077–82 (1989), Holm, et al., Cancer Research 49: 6365–8 (1989)). In cancer cells, camptothecin resistance can arise from a prolonged ability to arrest in G2 (Goldwasser, et al., Cancer Research 56: 4430–7 (1996), Dubrez, et al., Leukemia 9: 1013–24 (1995)), and from downregulation or mutation of Topo I (Arbuck, et al., Seminars in Hematology 35: 3–12 (1998), Larsen, et al., Biochimica et Biophysica Acta. 1400: 257–74 (1998)).
The clinical activity of the topoisomerase I poisons has been their ability to induce double-stranded breaks in the genome of dividing cells. Such poisons have been used to treat proliferative diseases such as cancer. However, currently available topoisomerase I poisons, such as camptothecin, are toxic only during S-phase of the cell cycle. What is needed in the art are new methods for inducing double-stranded breaks in DNA in all phases of the cell cycle by irreversibly trapping topoisomerase I-DNA complexes. Surprisingly, the present invention provides such methods and identifies compounds that are useful in such methods.